Signaling through cyclic AMP (cAMP) and its effector molecules, such as cAMP-dependent protein kinase (PKA) and exchange proteins activated by cAMP (Epac), regulates a variety of cellular functions including cell growth, proliferation, metabolism, survival and mobility, as well as insulin secretion in the case of pancreatic ? cells. The overall goal of our research is to elucidate the molecular mechanisms and functional roles of spatiotemporal compartmentalization in achieving high specificity in cAMP signaling. Aberrations in the cAMP signaling pathway have implications for clinical conditions such as obesity and type 2 diabetes mellitus, particularly in the context of ?-cell functions. A mechanistic understanding of cAMP signaling specificity is crucial to developing therapeutic strategies for these clinical conditions. The concept of spatial compartmentalization of cAMP effects was proposed 20 years ago, but only in recent years have innovative approaches to studying cAMP signaling in the cellular context become available to provide direct mechanistic evidence. However, despite these recent advances, there are still large gaps in our understanding about the mechanisms underlying the spatiotemporal regulation of cAMP and its effectors. Furthermore, little is known about how the signaling information encoded in the spatiotemporal patterns of activities is translated into specific functional responses. In our preliminary studies, we have developed fluorescent biosensors for monitoring Epac action, co-imaging approaches for tracking multiple signaling activities and a method for enzymatic manipulation of cAMP levels at subcellular locations. Furthermore, our recent studies have discovered an oscillatory circuit that consists of cAMP, PKA and Ca2+ in MIN6 ? cells. In the current proposal, building upon these preliminary findings, we will focus on these specific aims to test our central hypothesis that the activities of cAMP, PKA and Epac are spatiotemporally compartmentalized to specifically regulate functional effects of this pathway: 1) further develop molecular tools for measuring cAMP, PKA and Epac dynamics; 2) elucidate the regulatory mechanisms and functional roles of the oscillatory circuit in MIN6 b cells.